Intensive computer use has been associated with musculoskeletal disorders (MSDs). Although the underlying mechanisms are still not fully understood, muscle fatigue is thought to be a contributing factor. Previous studies have shown that keystroke durations are related to muscle twitch durations and may be used as a surrogate measure of muscle fatigue. Software tools have been developed to measure keystroke durations; however, the accuracy of these programs may be influenced by the computer and/or the operating system (OS). Keystrokes were collected from six subjects and analyzed to determine whether there were any differences in keystroke durations measured by an OS-dependant software program and keystrokes collected directly from the keyboard using a USB analyzer (gold standard). The results demonstrated that the OS-dependant software program underestimated keystroke durations by 3.8 ms (103.5 vs. 107.3 ms; p < 0.0001) but keystroke durations at the individual level were highly correlated between the two systems (R(2) = 0.997). Despite the small differences, the high correlation between systems indicated that the software program could be used to collect keystroke durations.